Compositions of morselized umbilical cord and/or amniotic membrane and methods of use thereof

ABSTRACT

The invention relates generally to the fields of biology and life sciences. More particularly, the invention relates to compositions and methods for modulating cellular physiology and pathological processing using a combination of compounds that can be found in morselized amniotic membrane tissue and morselized umbilical cord tissue preparations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/109,483 filed on Jan. 29, 2015 entitled “COMPOSITIONS AND METHODS”and U.S. Provisional Application No. 62/007,167 filed on Jun. 3, 2014entitled “COMPOSITIONS AND METHODS”, each of which is herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to the fields of biology and healthsciences. More particularly, the invention relates to compositions andmethods for modulating cellular physiology and pathological processingusing a combination of compounds that can be found in morselizedamniotic membrane tissue and morselized umbilical cord tissuepreparations.

BACKGROUND OF THE INVENTION

The placenta is a temporary organ that surrounds the fetus duringgestation. The placenta allows for transport of gases and nutrients, andalso provides other metabolic and endocrine functions. The placenta iscomposed of several tissue types. The umbilical cord (UC) connects theplacenta to the fetus, and transports oxygen to the fetus. The umbilicalcord has two arteries and a vein. Wharton's jelly, a specializedgelatinous connective tissue material, is within the umbilical cord andprotects and insulates the umbilical arteries and vein. The outermostlayer of the amniotic sac is known as the “chorion.” Much of theplacental disc is composed of chorionic villi, which are extensions ofthe chorionic villous tree. Through these structures, fetal nutritionexchange occurs. The amniotic membrane (AM) is an avascular membranoussac that is filled with amniotic fluid. This membrane is the innermostmembrane surrounding a fetus in the amniotic cavity. This tissueconsists of an epithelial layer and a subadjacent avascular stromallayer.

The umbilical cord (UC) and amniotic membrane (AM) are rich in stemcells and the resulting morselized umbilical cord and amniotic membranecompositions will therefore meet an unfilled need in the fields of woundcare and tissue regeneration.

SUMMARY OF THE INVENTION

Described herein are compositions of morselized amniotic membrane tissueand morselized umbilical cord tissue (that is, compositions that areprepared from amniotic membrane materials, including the amnioticmembrane, umbilical cord, amniotic stroma and amniotic jelly, amnioticfluid and Wharton's jelly). In some embodiments, at least one componentof the compositions are obtained from amniotic membrane tissue orumbilical cord tissue preparations. Also described herein arecompositions in which at least one component of the composition isobtained from human placenta, amniotic fluid and chorion. Also describedherein are methods for preparing any of the aforementioned compositionsand preparations. Also described herein are methods for storing andpreserving any of the aforementioned compositions and preparations. Alsodescribed herein are methods for using any of the aforementionedcompositions and preparations, including preservative methods, cellculture methods, tissue culture methods, therapeutic methods,prophylactic methods and cosmetic methods. Also described herein areapparatuses comprising compositions of morselized amniotic membranetissue and morselized umbilical cord tissue on an inert support. Alsodescribed herein are methods for preparing the aforementionedapparatuses. Also described herein are methods for using any of theaforementioned apparatuses, including preservative methods, cell culturemethods, tissue culture methods, therapeutic methods, prophylacticmethods and cosmetic methods.

Various umbilical cord and amniotic membrane compositions exert a numberof physiologically significant effects in mammalian cells and intactmammalian tissues. Such effects include suppressing TGF β signaling,increasing apoptosis of macrophages, decreasing cellular proliferationof, decreasing cellular migration of, and increasing apoptosis ofvascular endothelial cells, protecting corneal and limbal epithelialcells and keratocytes from apoptosis induced by storage or by dispasetreatment, and decreasing inflammation in tissues. Additionally themorselized umbilical cord and amniotic membrane compositions describedherein can be in liquid, semi-liquid or lyophilized form.

Although compositions, materials, and methods similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, suitable preparations, methods and materials aredescribed herein. All publications mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions will control. In addition, theparticular embodiments discussed below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1—Depiction of Morselized umbilical cord and amniotic membranecomposition on inert support.

DETAILED DESCRIPTION Certain Definitions

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The term “morsel” as used herein, refers to particles of tissue rangingin size from about 0.1 mm to about 1.0 cm in length, width, or thicknessthat have been obtained from a larger piece of tissue. A “morsel” asdescribed herein, retains the characteristics of the tissue from whichit was obtained and upon inspection is identifiable as said tissue.

The terms “morselized”, “morselizing” and “morselization” refer toactions involving the “morsels” of the present application.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition including a compound as disclosed herein required to providea clinically significant decrease in disease symptoms without undueadverse side effects. An appropriate “effective amount” in anyindividual case may be determined using techniques, such as a doseescalation study. The term “therapeutically effective amount” includes,for example, a prophylactically effective amount. An “effective amount”of a compound disclosed herein, is an amount effective to achieve adesired effect or therapeutic improvement without undue adverse sideeffects. It is understood that “an effective amount” or “atherapeutically effective amount” can vary from subject to subject, dueto variation in metabolism of the composition, age, weight, generalcondition of the subject, the condition being treated, the severity ofthe condition being treated, and the judgment of the prescribingphysician.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “kit” and “article of manufacture” are used as synonyms.

By “pharmaceutically acceptable,” as used herein, refers to a material,such as a carrier or diluent, which does not abrogate the biologicalactivity or properties of the compound, and is relatively nontoxic,i.e., the material may be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. the umbilical cord and amniotic membrane compositionsdescribed herein and a co-agent, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. theumbilical cord and amniotic membrane compositions described herein and aco-agent, are administered to a patient as separate entities eithersimultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

The term “protein” as used herein can be the full length polypeptide, ora fragment or segment of a polypeptide, and can encompass a stretch ofamino acid residues of at least about 8 amino acids, generally at least10 amino acids, more generally at least 20 amino acids, often at least30 amino acids, more often at least 50 amino acids or more of the fulllength polypeptide.

The term “inert support” as used herein refers to a biocompatiblematerial capable of being applied to a tissue of a patient in need oftreatment. Such materials may take the form of sheets. Further, suchmaterials may take the form of mesh sheets. Additionally, such materialsmay be manufactured from any biocompatible compound such as, forexample, collagen; hydrogels; polyethylene glycols;poly(lactic-co-glycolic acids); keratin; hydrophilic polyurethanes; andhydrocolloids

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Compositions

Described herein are compositions that exert a number of physiologicallysignificant effects in mammalian cells. intact mammalian tissues andmammalian patients in need thereof. The compositions comprise at leastone of two components: morselized amniotic membrane tissue andmorselized umbilical cord tissue.

Any or all of the components of the compositions described herein can beprepared from a human amniotic material, including human amniotic jellypreparations and extracts (as described herein), human amniotic membranepreparations and extracts (as described herein), and human amnioticstroma preparations and extracts (as described herein) or a humanumbilical cord material (as described herein) including human Wharton'sjelly preparations and extracts (as described herein).

These two components can suppress TGF β promoter activity; increaseapoptosis in macrophages; decrease proliferation, decrease migration,and increase apoptosis of human vascular endothelial cells; decreaseviability of human fibroblasts; decrease inflammation; and preventapoptosis of epithelial cells exposed to storage and injury.

These components can be obtained from any suitable source. For example,at least one of the components can be obtained from human tissues, suchas amniotic membrane, amniotic jelly, amniotic stroma, amniotic fluid,or a combination thereof. At least one of the components can be obtainedfrom commercial sources. At least one of the components can be isolatedfrom a transgenic organism. The protein sequences can have a similarityof at least 90%, 93%, 95%, 97%, 99% or 99.5% to the human proteinsequence. The components can be purified, substantially purified,partially purified, or non-purified. The components can also be preparedfrom mammalian amniotic membrane tissues, as each of the components ispresent in amniotic membrane tissues.

Human placental material can be obtained, for example, from sources suchas Bio-Tissue, Inc. (Miami, Fla.) and Baptist Hospital (Miami, Fla.)(under IRB approval). The tissue is typically obtained in either a freshor frozen state. The tissue can be washed to remove excess storagebuffer, blood, or contaminants. The excess liquid can be removed, forexample, using a brief centrifugation step, or by other means. Thetissue can be frozen, using, for example, liquid nitrogen or othercooling means, to facilitate the subsequent homogenization. The sourceof the umbilical cord and amniotic membrane tissue can be a human.However, other sources of umbilical cord and amniotic membrane tissue,such as bovine or porcine umbilical cord and amniotic membrane tissue,can be used.

A mixture of amniotic membrane tissue and umbilical cord tissue in anyratio from 0.001:99.999 w/w % to 99.999:0.001 w/w % can be morselizedfrom either fresh or frozen tissue through the use of any morselizingtool known to one of skill in the art such as, for example, tissuegrinder, sonicator, bread beater, freezer/mill, blender, mortar/pestle,Roto-stator, kitchen chopper, grater, ruler and scalpel to yield morselsranging in size from about 0.1 mm to about 1.0 cm in length, width, orthickness. Optionally, the resulting morsels may be homogenized to yieldconsistently sized morsels. The resulting morsels may be either usedwet, partially dehydrated or essentially dehydrated by any means knownto one of skill in the art such as, for example, centrifuge orlyophilization. The resulting composition may be used immediately orstored for later use in any type of contained known to one of skill inthe art such as, for example, pouch, jar, bottle, tube, ampule andpre-filled syringe. Finally, the morsel composition may be sterilized byany method known to one of skill in the art such as, for example, γradiation.

The placenta can be used to prepare the composition. Umbilical cord andamniotic membrane preparations can include components or portionsextracted from intact placentas. If desired, certain components of theumbilical cord and amniotic membrane preparation can be isolated fromthe preparation at any time during the process. The preparation can bedried, if desired.

The tissue can be frozen prior to the morselizing process. The freezingstep can occur by any suitable cooling process. For example, the tissuecan be flash-frozen using liquid nitrogen. Alternatively, the materialcan be placed in an isopropanol/dry ice bath or can be flash-frozen inother coolants. Commercially available quick freezing processes can beused. Additionally, the material can be placed in a freezer and allowedto equilibrate to the storage temperature more slowly, rather than beingflash-frozen. The tissue can be stored at any desired temperature. Forexample, −20° C. or −80° C. or other temperatures can be used forstorage.

Morselizing the tissue while frozen, rather than morselizing the tissueprior to freezing, is one optional method for preparing the tissue.Alternatively, fresh, partially thawed, or thawed tissue can be used inthe morselizing step. The tissue (fresh, frozen, or thawed) can then besliced into pieces of a desired size with a suitable device, such as ascalpel, and homogenized with a homogenization device such as alaboratory blender, in a suitable solution. Exemplary solutions includebut are not limited to phosphate buffered saline (PBS), DMEM, NaClsolution, and water. The pH of the solution can be adjusted as needed.In some embodiments, the pH range is from about 5.5 or 6.0 to about 8.5.In some embodiments, the frozen tissue is morselized in a solutionhaving a pH of between about 6.3, about 6.6, or about 7.0 to about 7.4,about 7.6, or about 7.8.

Umbilical cord and amniotic membrane preparations can be in a liquid,suspension, or lyophilized forms. Antimicrobial agents such asantibiotics or anti-fungal agents may be added. The material can bepackaged and stored, for example, at room temperature, or for example,at −20° C. or −80° C. prior to use.

In some embodiments, the preparation is present as a dry formulation. Adry formulation can be stored in a smaller volume, and may not requirethe same low temperature storage requirements to keep the formulationfrom degrading over time. A dry formulation can be stored andreconstituted prior to use. The dry formulation can be prepared, forexample, by preparing the freeze-morselized umbilical cord and amnioticmembrane tissue as described herein, then removing at least a portion ofthe water in the composition. The excess water can be removed from thepreparation by any suitable means. An exemplary method of removing thewater is by use of lyophilization using a commercially availablelyophilizer or freeze-dryer. Suitable equipment can be found, forexample, through Virtis, Gardiner, N.Y.; FTS Systems, Stone Ridge, N.Y.;and SpeedVac (Savant Instruments Inc., Farmingdale, N.Y.). The amount ofwater that is removed can be from about 5%, 10%, 20%, 30% to about 60,70, 80, 90, 95 or 99% or more. In some embodiments, substantially all ofthe excess water is removed. The lyophilized composition can then bestored. The storage temperature can vary from less than about −196°C.-80° C., −50° C., or −20° C. to more than about 23° C. If desired, thecomposition can be characterized (weight, protein content, etc.) priorto storage.

The lyophilized composition can be reconstituted in a suitable solutionor buffer prior to use. Exemplary solutions include but are not limitedto PBS, DMEM, and BSS. The pH of the solution can be adjusted as needed.The concentration of the umbilical cord and amniotic membrane can bevaried as needed. In some procedures a more concentrated preparation isuseful, whereas in other procedures, a solution with a low concentrationof umbilical cord and amniotic membrane is useful. Additional compoundscan be added to the composition. Exemplary compounds that can be addedto the reconstituted formulation include but are not limited to pHmodifiers, buffers, collagen, hyaluronic acid (HA), antibiotics,surfactants, stabilizers, proteins, and the like. The lyophilizedumbilical cord and amniotic membrane composition can also be added to aprepared cream, ointment or lotion to result in the desiredconcentration.

Apparatuses:

Described herein are apparatuses that when contacted with a tissue of apatient in need of treatment exert a number of physiologicallysignificant effects in mammalian cells and intact mammalian tissues. Theapparatuses comprise at least one tissue selected from the groupconsisting of: morselized amniotic membrane tissue; morselized umbilicalcord tissue; and combinations thereof; and at least one inert support,wherein, the at least one tissue is dispersed upon at least one surfaceof the at least one inert support.

In certain embodiments, the at least one tissue comprises pieces ofamniotic membrane tissue from about 0.3 mm to about 1.0 cm in length,width and thickness. Further, in certain embodiments, the at least onetissue comprises pieces of umbilical cord tissue from about 0.3 mm toabout 1.0 cm in length, width and thickness. Finally, in certainembodiments, the at least one tissue may be present in any ratio fromabout 0.001:99.999 w/w % to about 99.999:0.001 w/w % of morselizedamniotic membrane tissue to morselized umbilical cord tissue,respectively.

In other embodiments, the at least one tissue has a reduced watercontent by weight percentage. In still other embodiments, the at leastone tissue has a water content by weight percentage greater than 20%.

In some embodiments, the apparatus is sterilized. In additionalembodiments, the sterilization is accomplished by gamma (γ) radiation.In some embodiments, the at least one inert support is a mesh sheethaving an average pore size from about 0.2 mm to about 0.9 cm.

In certain embodiments, the at least one inert support comprises amaterial selected from the group consisting of: collagen; hydrogels;keratin; hydrophilic polyurethane; polyethylene glycols;poly(lactic-co-glycolic acids); and hydrocolloids. Further, in certainembodiments, the natural biological activity of the morselized amnioticmembrane tissue and the morselized umbilical cord tissue issubstantially preserved for at least 15 days after initial procurement.Additionally, in certain embodiments, the apparatus isanti-inflammatory, anti-scarring, anti-angiogenic, anti-adhesion, orpromotes wound healing when contacted with an exogenous living cell. Insome embodiments substantially all red blood cells have been removedfrom the morselized amniotic membrane tissue and the morselizedumbilical cord tissue. In some embodiments, substantially all choriontissue has been removed from the morselized amniotic membrane tissue andthe morselized umbilical cord tissue. In some embodiments, at least somechorion tissue remains with the morselized amniotic membrane tissue andthe morselized umbilical cord tissue. In some embodiments, the at leastone tissue further comprises amniotic fluid

In other embodiments, the at least one tissue is cryopreserved,lyophilized, or a combination thereof. In some embodiments, the at leastone inert support has a surface area from about 6 cm² to about 200 cm².In some embodiments, the apparatus further comprises at least oneadditional type of cell selected from the group consisting of: limbalepithelial stem cells, keratocytes, human umbilical vein endothelialcells, mesenchymal stem cells, adipose-derived stem cells, endothelialstem cells and dental pulp stem cells. In some embodiments, the at leastone tissue is a morselized homogenate.

The following procedures represent illustrative methods for preparingthe umbilical cord and amniotic membrane compositions described and usedherein.

Preparation of Preserved Human Umbilical Cord and Amniotic Membrane:

Human placenta was collected at elective cesarean delivery (Heiligenhauset al., Invest Ophthalmol Vis Sci. 42:1969-1974, 2001, Lee and Tseng, AmJ Ophthalmol. 123:303-312, 1997, Prabhasawat et al., Ophthalmology,104:974-985, 1997, Tseng et al., Arch Ophthalmol. 116:431-441, 1998).The umbilical cord and amniotic membrane was flattened ontonitrocellulose paper (Hybond N+, Amersham, England), with the epitheliumsurface up. The umbilical cord and amniotic membrane samples were storedat −80° C. in DMEM/glycerol 1:2 (v/v) until use.

Umbilical Cord and Amniotic Membrane Compositions

Umbilical cord and amniotic membrane compositions can be formulated foradministration purposes as a non-solid dosage form, for example, bycombining with a delivery vehicle to create compositions such assolutions, drops, suspensions, pastes, sprays, ointments, oils,emulsions, aerosols, a coated bandage, a patch, creams, lotions, gels,and the like. The formulation used will depend upon the particularapplication. Gels are useful for administering the compositions becausethey allow better retention of the active ingredient at the site ofintroduction, allowing the active ingredient to exert its effect for alonger period of time before clearance of the active ingredient. Adescription of exemplary pharmaceutically acceptable carriers orvehicles and diluents, as well as pharmaceutical formulations, isprovided herein and can also be found in Remington's PharmaceuticalSciences, a standard text in this field, and in USP/NF.

Compositions may be formulated in a conventional manner using one ormore physiologically acceptable carriers including excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any of the well-knowntechniques, carriers, and excipients may be used as suitable and asunderstood in the art. A summary of pharmaceutical compositionsdescribed herein may be found, for example, in Remington: The Scienceand Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack PublishingCompany, 1995); Hoover, John E., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins; 1999), herein incorporated by referencein their entirety.

In certain embodiments, the compositions include a pharmaceuticallyacceptable diluent(s), excipient(s), or carrier(s). In addition, theumbilical cord and amniotic membrane compositions described herein canbe administered as compositions in which umbilical cord and amnioticmembrane compositions described herein are mixed with other activeingredients, as in combination therapy. In some embodiments, thecompositions may include other medicinal or pharmaceutical agents,carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, the compositions can also containother therapeutically effective substances.

A composition, as used herein, refers to a mixture of a umbilical cordand amniotic membrane compositions described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. The compositionfacilitates administration of the compound to an organism. In practicingthe methods of treatment or use provided herein, therapeuticallyeffective amounts of umbilical cord and amniotic membrane compositionsdescribed herein are administered to a mammal having a disease,disorder, or condition to be treated. In some embodiments, the mammal isa human. A therapeutically effective amount can vary widely depending onthe severity of the disease, the age and relative health of the subject,the potency of the compound used and other factors. The compounds can beused singly or in combination with one or more therapeutic agents ascomponents of mixtures.

Topical Formulations

Formulations of the umbilical cord and amniotic membrane compositionsdescribed herein include those suitable for topical administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredients which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated and the particular mode of administration.

Suspensions may contain suspending agents as, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

Typical compositions described herein include a wide variety of physicalforms. These include, but are not limited to, solutions, lotions,creams, oils, gels, sticks, sprays, ointments, balms, shampoo, andpastes. Generally, such carrier systems can be described as beingsolutions, emulsions, gels, solids, and aerosols. The compositions maybe applied topically to the skin, or may be applied in the form of atransdermal delivery device, such as a microneedle, a patch, bandage, orgauze pad known in the art.

The ointments, pastes, creams and gels may contain, in addition to theumbilical cord and amniotic membrane compositions described herein,excipients, such as animal and vegetable fats, oils, waxes, paraffins,starch, tragacanth, cellulose derivatives, polyethylene glycols,silicones, bentonites, silicic acid, talc and zinc oxide, or mixturesthereof.

Powders and sprays can contain, in addition to the umbilical cord andamniotic membrane compositions described herein, excipients such aslactose, talc, silicic acid, aluminum hydroxide, calcium silicates andpolyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Solvents are generally employed in the preparation of suitable topicalcompositions. Such solvents can either be aqueous or organic based. Thesolvent must be capable of having dispersed or dissolved therein theactive ingredients while not being irritating to the animal beingtreated. Water forms the basis for all aqueous solvents, while suitableorganic solvents include propylene glycol, butylene glycol, polyethyleneglycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitolesters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol, andmixtures thereof. Solvents can be included in the overall composition inamounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. Insome embodiments, the compositions are produced in the form of anemollient-containing composition. A wide variety of suitable emollientsare known and may be used herein.

In some embodiments, the compositions are formulated as lotionscontaining from about 0.01% to 10% of the umbilical cord and amnioticmembrane compositions described herein. In other embodiments, thecompositions are formulated in a solution carrier system as a cream. Acream composition would preferably comprise from about 0.1% to 15% andpreferably from 1% to 5% of the umbilical cord and amniotic membranecompositions described herein. Lotions and creams can be formulated asemulsions as well as solutions. The compositions may also beadministered in liquid form, including in the form of liposomessuspended in liquid, as in the different type of sprays available inthis industry.

In other embodiments, the active ingredients are formulated asointments. Suitable ointments may comprise simple bases of animal orvegetable oils, or semi-solid hydrocarbons (oleaginous). Suitableointments may also comprise absorption ointment bases which absorb waterto form emulsions. Ointment carriers may also be water soluble. Anointment may comprise from 1% to 99% of an emollient plus to about 0.1%to 99% of a thickening agent.

The proportion of the umbilical cord and amniotic membrane compositionsdescribed herein in the compositions can vary from between about 0.01wt. % to about 100 wt. %, more preferably from about 0.1 wt. % to about99.9 wt. %, and especially from about 1.0 wt. % to about 99.0 wt. %.

“Carriers” or “vehicles” preferably refer to carrier materials suitablefor topical administration and include any such materials known in theart, such as any liquid, gel solvent, liquid diluent, solubilizer, orthe like, which is non-toxic, and which does not interact with othercomponents of the composition in a deleterious manner. Examples ofsuitable carriers for use herein include water, silicone, liquid sugars,waxes, oils, petroleum jelly, and a variety of other materials.

In some embodiments, the carrier or vehicle includes one or moresolvents, oils, surfactants, humectants, thickening agents,antioxidants, chelating agents, buffers, and preservatives.

Examples of solvents include C2-C10 alcohols, such as hexanol,cyclohexanol, benzyl alcohol, 1,2-butanediol, glycerol, and amylalcohol; C5-C10 hydrocarbons such as n-hexane, cyclohexane, andethylbenzene; C4-C10 aldehydes and ketones, such as heptylaldehyde,cyclohexanone, and benzylaldehyde; C4-C10 esters, such as amyl acetateand benzyl propionate; ethereal oils, such as oil of eucalyptus, oil ofrue, cumin oil, limonene, thymol, and 1-pinene; halogenated hydrocarbonshaving 2-8 carbon atoms, such as 1-chlorohexane, 1-bromohexane, andchlorocyclohexane.

Examples of oils comprise fats and oils such as olive oil andhydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons suchas liquid paraffin, ceresin, and squalane; fatty acids such as stearicacid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol,lanolin alcohol, and hexadecanol; and esters such as isopropylmyristate, isopropyl palmitate and butyl stearate.

Examples of surfactants include anionic surfactants such as sodiumstearate, sodium cetyl sulfate, polyoxyethylene laurylether phosphate,sodium N-acyl glutamate; cationic surfactants such asstearyldimethylbenzylammonium chloride and stearyltrimethylammoniumchloride; ampholytic surfactants such as alkylaminoethylglycinehydrochloride solutions and lecithin; and nonionic surfactants such asglycerin monostearate, sorbitan monostearate, sucrose fatty acid esters,propylene glycol monostearate, polyoxyethylene oleylether, polyethyleneglycol monostearate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropyleneglycol (e.g., the materials sold under the trademark “Pluronic”),polyoxyethylene castor oil, and polyoxyethylene lanolin.

Examples of humectants include glycerin, 1,3-butylene glycol, andpropylene glycol; examples of thickening agents include xanthan gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethyleneglycol and sodium carboxymethyl cellulose; examples of antioxidantscomprise butylated hydroxytoluene, butylated hydroxyanisole, propylgallate, citric acid and ethoxyquin; examples of chelating agentsinclude disodium edetate and ethanehydroxy diphosphate; examples ofbuffers comprise citric acid, sodium citrate, boric acid, borax, anddisodium hydrogen phosphate; and examples of preservatives are methylparahydroxybenzoate, ethyl parahydroxybenzoate, dehydroacetic acid,salicylic acid and benzoic acid.

In certain embodiments, the carrier/vehicle is composed of the foregoingmaterials to achieve a controlled occlusion of the skin, therebyresulting in optimal enhancement of biologically active moietypenetration across the skin with minimal skin irritation. In certainembodiments, the carrier/vehicle may include a dispersing agent thataids in maintaining a particulate phase of the active ingredientsdispersed in the continuous phase. In other embodiments, non-ionicexcipients, such as lauric alcohol, propylene glycol monolaurate,myristyl lactate, lauryl lactate, or the like, facilitate dispersion.

The rate of biologically active moiety delivery across a dermal surfacecan be increased by transdermal delivery enhancers. Suitable transdermaldelivery enhancers include proton-accepting solvents such asdimethylsulfoxide and dimethylacetamide. Other suitable transdermaldelivery enhancers include 2-pyrrolidine, N,N-diethyl-m-toluamide,1-dodecylazacycloheptan-2-one, N,N-dimethylformamide,N-methyl-2-pyrrolidine, terpenes, surfactants, and calciumthioglycolate.

Suitable dermal penetration enhancers include 1-5 carbon fatty acidesters of para-aminobenzoic acid, isopropyl palmitate, isopropylmyristate, ethanol, isobutyl alcohol, isobutyl alcohol, stearyl alcohol,glycerol, 2-pyrrolidone, urea, propylene glycol, oleic acid, palmiticacid, dimethyl sulfoxide, N,N-dimethyl acetamide, N,N-dimethylformamide, 2-pyrrolidone, 1-methyl-2-pyrrolidone,5-methyl-2-pyrrolidone, 1,5-dimethyl-2-pyrrolidone,1-ethyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid,N,N-dimethyl-m-toluamide, urea, ethyl acetate,1-dodecylazacycloheptan-2-one, oleic acid, imidazoline, butylurea, andpyrrolidone carboxylic acid esters.

Wetting agents, emulsifiers, surfactants, and lubricants, such as sodiumlauryl sulfate and magnesium stearate, as well as coloring agents,release agents, coating agents, sweetening, flavoring, and perfumingagents, preservatives and antioxidants can also be present in thecompositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite,and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

As appropriate compositions for topical application there may be citedall compositions usually employed for topically administeringtherapeutics, e.g., creams, jellies, dressings, shampoos, tinctures,pastes, ointments, salves, powders, liquid or semiliquid formulations,and the like. Application of said compositions may be by aerosol, e.g.,with a propellant such as nitrogen carbon dioxide, a freon, or without apropellant such as a pump spray, drops, lotions, or a semisolid such asa thickened composition which can be applied by a swab. In particularcompositions, semisolid compositions such as salves, creams, pastes,jellies, ointments, and the like will conveniently be used.

Methods of Dosing and Treatment Regimens

The compositions can be administered by any suitable technique.Typically, the compositions will be administered directly to a targetsite (e.g., wound. dermal ulcer, skin).]. If delivery of umbilical cordand amniotic membrane preparations to the skin is desired, topicaladministration can be used.

The compositions containing the umbilical cord and amniotic membranecompositions described herein can be administered for [therapeutictreatments. In therapeutic applications, the compositions areadministered to a patient already suffering from a disease or condition,in an amount sufficient to cure or at least partially arrest thesymptoms of the disease or condition. Amounts effective for this usewill depend on the severity and course of the disease or condition,previous therapy, the patient's health status, weight, and response tothe drugs, and the judgment of the treating physician. It is consideredwell within the skill of the art for one to determine suchtherapeutically effective amounts by routine experimentation (including,but not limited to, a dose escalation clinical trial).

Combination Treatments

The umbilical cord and amniotic membrane compositions and methodsdescribed herein may also be used in conjunction with other well-knowntherapeutic reagents that are selected for their particular usefulnessagainst the condition that is being treated. In general, thecompositions described herein and, in embodiments where combinationaltherapy is employed, other agents do not have to be administered in thesame composition, and may, because of different physical and chemicalcharacteristics, have to be administered by different routes. Thedetermination of the mode of administration and the advisability ofadministration, where possible, in the same composition, is well withinthe knowledge of the skilled clinician. The initial administration canbe made according to established protocols known in the art, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration can be modified by the skilled clinician.

The particular choice of compounds used will depend upon the diagnosisof the attending physicians and their judgment of the condition of thepatient and the appropriate treatment protocol. The compounds may beadministered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disease, disorder, or condition, thecondition of the patient, and the actual choice of compounds used. Thedetermination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the patient.

It is known to those of skill in the art that therapeutically-effectivedosages can vary when the drugs are used in treatment combinations.Methods for experimentally determining therapeutically-effective dosagesof drugs and other agents for use in combination treatment regimens aredescribed in the literature. For example, the use of metronomic dosing,i.e., providing more frequent, lower doses in order to minimize toxicside effects, has been described extensively in the literature.Combination treatment further includes periodic treatments that startand stop at various times to assist with the clinical management of thepatient.

For combination therapies described herein, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disease orcondition being treated and so forth. In addition, when co-administeredwith one or more biologically active agents, the compound providedherein may be administered either simultaneously with the biologicallyactive agent(s), or sequentially. If administered sequentially, theattending physician will decide on the appropriate sequence ofadministering protein in combination with the biologically activeagent(s).

In any case, the multiple therapeutic agents may be administered in anyorder or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents; the use of multiple therapeutic combinations arealso envisioned.

It is understood that the dosage regimen to treat or ameliorate thecondition(s) for which relief is sought, can be modified in accordancewith a variety of factors. These factors include the disorder from whichthe subject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, the dosage regimen actually employed canvary widely and therefore can deviate from the dosage regimens set forthherein.

The pharmaceutical agents which make up the combination therapydisclosed herein may be a combined dosage form or in separate dosageforms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy may also beadministered sequentially, with either therapeutic compound beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen may call for sequential administrationof the active agents or spaced-apart administration of the separateactive agents. The time period between the multiple administration stepsmay range from, a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent. Circadian variation of the target moleculeconcentration may also determine the optimal dose interval.

In addition, the umbilical cord and amniotic membrane compositionsdescribed herein also may be used in combination with procedures thatmay provide additional or synergistic benefit to the patient. By way ofexample only, patients are expected to find therapeutic and/orprophylactic benefit in the methods described herein, whereincomposition of a compound disclosed herein and/or combinations withother therapeutics are combined with genetic testing to determinewhether that individual is a carrier of a mutant gene that is known tobe correlated with certain diseases or conditions.

Kits/Articles of Manufacture

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits can includea carrier, package, or container that is compartmentalized to receiveone or more containers such as vials, tubes, and the like, each of thecontainer(s) including one of the separate elements to be used in amethod described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. A wide array of formulations of thecompounds and compositions provided herein are contemplated as are avariety of treatments for any disease, disorder, or condition.

For example, the container(s) can include one or more umbilical cord andamniotic membrane compositions described herein, optionally in acomposition or in combination with another agent as disclosed herein.The container(s) optionally have a sterile access port (for example thecontainer can be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). Such kits optionallycomprising a compound with an identifying description or label orinstructions relating to its use in the methods described herein.

A kit will typically include one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of the umbilical cord and amniotic membranecompositions described herein. Non-limiting examples of such materialsinclude, but not limited to, buffers, diluents, filters, needles,syringes; carrier, package, container, vial and/or tube labels listingcontents and/or instructions for use, and package inserts withinstructions for use. A set of instructions will also typically beincluded.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein.

In certain embodiments, the compositions or apparatuses can be presentedin a pack or dispenser device which can contain one or more unit dosageforms containing a compound provided herein. The pack can for examplecontain metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.The pack or dispenser can also be accompanied with a notice associatedwith the container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of the drugfor human or veterinary administration. Such notice, for example, can bethe labeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. Compositionscontaining a compound provided herein formulated in a compatible carriercan also be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition.

Methods of Treatment

The umbilical cord and amniotic membrane compositions and apparatusesdescribed herein have many uses including research and clinicalapplications. Based on the results described herein, the umbilical cordand amniotic membrane compositions described herein can be applied totissues, cells or patients in need thereof to achieve a desiredmodulation of physiology.

Specifically, the umbilical cord and amniotic membrane compositions andapparatuses described herein may be useful for treating a patientsuffering from a disease or disorder selected from the group consistingof: skin wounds and repairs (e.g. burns, necrosis, skin ulcers andvenous ulcers), ocular wounds and repairs (e.g. glaucoma, ocular ulcers,corneal ulcers, conjunctival, scleral and lid and orbital rimreconstruction), cell transplant, coronary wounds and repairs (e.g.coronary artery bypass, heart valve repair/replacement, vein repair andartery repair), nerve repair, spinal repair, psoriasis, plaque psoriasisand rheumatoid arthritis.

Additionally, the umbilical cord and amniotic membrane compositions andapparatuses described may used as a dermal filler for a patient in needthereof.

EXAMPLES

A umbilical cord and amniotic membrane composition as described herein,was slowly poured over 300 μm mesh inert support with the aid of avacuum filter system. Particles in the desired size range remained uponthe inert support to form an apparatus of the present application whichmay then be used to treat a patient in need thereof.

While it is apparent that the embodiments of the invention hereindisclosed are well suited to fulfill the objectives stated above, itwill be appreciated that numerous modifications and other embodimentsmay be implemented by those skilled in the art, and it is intended thatthe appended claims cover all such modifications and embodiments thatfall within the true spirit and scope of the present invention.

A number of references have been cited and the entire disclosures ofwhich are incorporated herein by reference.

What is claimed is:
 1. A method for treating an ocular wound orrepairing damaged ocular tissue in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of a gel composition comprising: a) a therapeutically effectiveamount of i) morselized placental amniotic membrane and ii) morselizedumbilical cord; and b) a pharmaceutically-acceptable excipient; whereinthe morselized placental amniotic membrane and the morselized umbilicalcord have a particle size of between about 0.1 mm to about 1.0 cm inlength, width, and thickness.
 2. The method according to claim 1,wherein the individual has a glaucoma-induced ocular wound,glaucoma-induced ocular tissue damage, an ocular ulcer, a corneal ulcer,a conjunctival reconstruction, a scleral reconstruction, a lidreconstruction, or an orbital rim reconstruction.
 3. The methodaccording to claim 1, wherein the composition has a reduced watercontent by weight percentage compared to the morselized placentalamniotic membrane or the morselized umbilical cord before morselization.4. The method according to claim 1, wherein the composition has a watercontent greater than 20% by weight percentage compared to the morselizedplacental amniotic membrane or the morselized umbilical cord beforemorselization.
 5. The method according to claim 1, wherein thecomposition is sterilized.
 6. The method according to claim 5, whereinthe composition is sterilized by gamma (γ) radiation.
 7. The methodaccording claim 1, wherein the morselized placental amniotic membraneand the morselized umbilical cord have a biological activity that issubstantially preserved for at least 15 days after initial procurement.8. The method according to claim 1, wherein the composition isanti-inflammatory, anti-scarring, anti-angiogenic, anti-adhesion, orpromotes wound healing.
 9. The method according to claim 1, wherein themorselized placental amniotic membrane and the morselized umbilical cordsubstantially do not comprise red blood cells.
 10. The method accordingto claim 1, wherein the morselized placental amniotic membranesubstantially does not comprise chorion.
 11. The method according toclaim 1, wherein the morselized placental amniotic membrane compriseschorion.
 12. The method according to claim 1, wherein the compositionfurther comprises amniotic fluid.
 13. The method according to claim 1,wherein the morselized placental amniotic membrane, morselized umbilicalcord or combination thereof is cryopreserved, lyophilized, or acombination thereof.
 14. The method according to claim 1, wherein thecomposition further comprises at least one pharmaceutically acceptablecarrier or diluent selected from the group consisting of: acacia,gelatin, colloidal silicon dioxide, calcium glycerophosphate, calciumlactate, maltodextrin, glycerine, magnesium silicate,polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodiumcaseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodiumchloride, tricalcium phosphate, dipotassium phosphate, cellulose andcellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasiccalcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate,calcium phosphate; anhydrous lactose, spray-dried lactose, compressiblesugar, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, confectioner's sugar; monobasic calciumsulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; sodium chloride,inositol, and bentonite.
 15. The method according to claim 1, whereinthe morselized placental amniotic membrane, morselized umbilical cord orcombination thereof is a homogenate.
 16. A method for treating an ocularwound or repairing a damaged ocular tissue in an individual in needthereof, comprising: applying to the individual an apparatus comprising(a) an inert support, and (b) a gel composition comprising morselizedplacental amniotic membrane, morselized umbilical cord, or a combinationthereof; wherein the morselized placental amniotic membrane and themorselized umbilical cord have a particle size of between about 0.1 mmto about 1.0 cm in length, width, and thickness.
 17. The methodaccording to claim 16, wherein the individual has a glaucoma-inducedocular wound, glaucoma-induced ocular tissue damage, an ocular ulcer, acorneal ulcer, a conjunctival reconstruction, a scleral reconstruction,a lid reconstruction or an orbital rim reconstruction.
 18. The methodaccording to claim 16, wherein the morselized placental amnioticmembrane, morselized umbilical cord or combination thereof has a reducedwater content by weight percentage compared to the morselized placentalamniotic membrane or the morselized umbilical cord before morselization.19. The method according to claim 18, wherein the morselized placentalamniotic membrane, morselized umbilical cord or combination thereof hasa water content greater than 20% by weight percentage.
 20. The methodaccording to claim 16, wherein the apparatus is sterilized.
 21. Themethod according to claim 20, wherein the apparatus is sterilized bygamma (γ) radiation.
 22. The method according to claim 16, wherein theinert support is a mesh sheet with an average pore size of about 0.2 mmto about 0.9 cm.
 23. The method according to claim 22, wherein the inertsupport comprises a material selected from the group consisting of:collagen; hydrogels; keratin; hydrophilic polyurethane; polyethyleneglycols; poly(lactic-co-glycolic acids); and hydrocolloids.
 24. Themethod according claim 16, wherein the morselized placental amnioticmembrane and the morselized umbilical cord have a biological activitythat is substantially preserved for at least 15 days after initialprocurement.
 25. The method according to claim 16, wherein the apparatusis anti-inflammatory, anti-scarring, anti-angiogenic, anti-adhesion, orpromotes wound healing.
 26. The method according to claim 16, whereinthe morselized placental amniotic membrane and the morselized umbilicalcord substantially do not comprise red blood cells.
 27. The methodaccording to claim 16, wherein the morselized placental amnioticmembrane substantially does not comprise chorion.
 28. The methodaccording to claim 16, wherein the morselized placental amnioticmembrane further comprises chorion.
 29. The method according to claim16, wherein the apparatus further comprises amniotic fluid.
 30. Themethod according to claim 16, wherein the morselized placental amnioticmembrane, morselized umbilical cord or combination thereof iscryopreserved, lyophilized, or a combination thereof.
 31. The methodaccording to claim 16, wherein the morselized placental amnioticmembrane, morselized umbilical cord or combination thereof is ahomogenate.